Cognate and noncognate metal ion coordination in metal-specific metallothioneins: the Helix pomatia system as a model.

The Helix pomatia metallothionein (MT) system, namely, its two highly specific forms, HpCdMT and HpCuMT, has offered once again an optimum model to study metal-protein specificity. The present work investigates the most unexplored aspect of the coordination behavior of MT polypeptides with respect to either cognate or noncognate metal ions, as opposed to the standard studies of cognate metal ion coordination. To this end, we analyzed the in vivo synthesis of the corresponding complexes with their noncognate metals, and we performed a detailed spectroscopic and spectrometric study of the Zn(2+)/Cd(2+) and Zn(2+)/Cu(+) in vitro replacement reactions on the initial Zn-HpMT species. An HpCuMTAla site-directed mutant, exhibiting differential Cu(+)-binding abilities in vivo, was also included in this study. We demonstrate that when an MT binds its cognate metal, it yields well-folded complexes of limited stoichiometry, representative of minimal-energy conformations. In contrast, the incorporation of noncognate metal ions is better attributed to an unspecific reaction of cysteinic thiolate groups with metal ions, which is dependent on their concentration in the surrounding milieu, where no minimal-energy structure is reached, and otherwise, the MT peptide acts as a multidentate ligand that will bind metal ions until its capacity has been saturated. Additionally, we suggest that previous binding of an MT polypeptide with its noncognate metal ion (e.g., binding of Zn(2+) to the HpCuMT isoform) may preclude the correct folding of the complex with its cognate metal ion.

Shaping mechanisms of metal specificity in a family of metazoan metallothioneins: evolutionary differentiation of mollusc metallothioneins.

BACKGROUND: The degree of metal binding specificity in metalloproteins such as metallothioneins (MTs) can be crucial for their functional accuracy. Unlike most other animal species, pulmonate molluscs possess homometallic MT isoforms loaded with Cu(+) or Cd(2+). They have, so far, been obtained as native metal-MT complexes from snail tissues, where they are involved in the metabolism of the metal ion species bound to the respective isoform. However, it has not as yet been discerned if their specific metal occupation is the result of a rigid control of metal availability, or isoform expression programming in the hosting tissues or of structural differences of the respective peptides determining the coordinative options for the different metal ions. In this study, the Roman snail (Helix pomatia) Cu-loaded and Cd-loaded isoforms (HpCuMT and HpCdMT) were used as model molecules in order to elucidate the biochemical and evolutionary mechanisms permitting pulmonate MTs to achieve specificity for their cognate metal ion. RESULTS: HpCuMT and HpCdMT were recombinantly synthesized in the presence of Cd(2+), Zn(2+) or Cu(2+) and corresponding metal complexes analysed by electrospray mass spectrometry and circular dichroism (CD) and ultra violet-visible (UV-Vis) spectrophotometry. Both MT isoforms were only able to form unique, homometallic and stable complexes (Cd(6)-HpCdMT and Cu(12)-HpCuMT) with their cognate metal ions. Yeast complementation assays demonstrated that the two isoforms assumed metal-specific functions, in agreement with their binding preferences, in heterologous eukaryotic environments. In the snail organism, the functional metal specificity of HpCdMT and HpCuMT was contributed by metal-specific transcription programming and cell-specific expression. Sequence elucidation and phylogenetic analysis of MT isoforms from a number of snail species revealed that they possess an unspecific and two metal-specific MT isoforms, whose metal specificity was achieved exclusively by evolutionary modulation of non-cysteine amino acid positions. CONCLUSION: The Roman snail HpCdMT and HpCuMT isoforms can thus be regarded as prototypes of isoform families that evolved genuine metal-specificity within pulmonate molluscs. Diversification into these isoforms may have been initiated by gene duplication, followed by speciation and selection towards opposite needs for protecting copper-dominated metabolic pathways from nonessential cadmium. The mechanisms enabling these proteins to be metal-specific could also be relevant for other metalloproteins.

Lack of DNA helicase Pif1 disrupts zinc and iron homoeostasis in yeast.

The Saccharomyces cerevisiae gene PIF1 encodes a conserved eukaryotic DNA helicase required for both mitochondrial and nuclear DNA integrity. Our previous work revealed that a pif1Δ strain is tolerant to zinc overload. In the present study we demonstrate that this effect is independent of the Pif1 helicase activity and is only observed when the protein is absent from the mitochondria. pif1Δ cells accumulate abnormal amounts of mitochondrial zinc and iron. Transcriptional profiling reveals that pif1Δ cells under standard growth conditions overexpress aconitase-related genes. When exposed to zinc, pif1Δ cells show lower induction of genes encoding iron (siderophores) transporters and higher expression of genes related to oxidative stress responses than wild-type cells. Coincidently, pif1Δ mutants are less prone to zinc-induced oxidative stress and display a higher reduced/oxidized glutathione ratio. Strikingly, although pif1Δ cells contain normal amounts of the Aco1 (yeast aconitase) protein, they completely lack aconitase activity. Loss of Aco1 activity is also observed when the cell expresses a non-mitochondrially targeted form of Pif1. We postulate that lack of Pif1 forces aconitase to play its DNA protective role as a nucleoid protein and that this triggers a domino effect on iron homoeostasis resulting in increased zinc tolerance.

Evidence of native metal-S(2-)-metallothionein complexes confirmed by the analysis of Cup1 divalent-metal-ion binding properties.

It has previously been shown that recombinant synthesis, under metal-supplemented conditions, of diverse metallothioneins (MTs) results in the recovery of a subpopulation of S(2-)-containing complexes in addition to the S(2-)-devoid canonical metal-MT species. Further significance of this finding has remained veiled by the possibility of it being a mere consequence of synthesis in a heterologous bacterial system. Herein, we present definitive evidence that S(2-) ligands are also constituents of native metal-MT complexes. Because, although practically universal, the highest S(2-) content is incorporated by copper-thioneins when coordinating divalent metal ions, we adapted the Saccharomyces cerevisiae Cup1 protein, which is the most paradigmatic copper-thionein, as an experimental model. Most significantly, native Cd-Cup1 complexes were purified and fully spectroscopically and spectrometrically characterized from the 301N mutant yeast strain, which allows Cup1 synthesis even in the absence of copper. These results undoubtedly revealed the presence of a Cd-S(2-)-Cup1 species in native preparations, which were only recovered when carefully avoiding the use of ion-exchange chromatography in the purification protocol. Furthermore, complete analysis of recombinant (Escherichia coli) Zn-Cup1, Cd-Cup1, and Cu-Cup1 and those complexes that result from Zn/Cd and Zn/Cu replacements in vitro and acidification/renaturalization processes yielded a comprehensive and comparative overview of the metal-binding abilities of Cup1. Overall, we consider the main conclusions of this study to go beyond the mere study of the particular Cup1 MT, so that they should be considered to delineate a new point of view on the interaction between copper-thioneins and divalent metal ions, still an unexplored aspect in MT research.

The Saccharomyces cerevisiae Crs5 Metallothionein metal-binding abilities and its role in the response to zinc overload.

Crs5 is a Saccharomyces cerevisiae Metallothionein (MT), non-homologous to the paradigmatic Cu-thionein Cup1. Although considered a secondary copper-resistance agent, we show here that it determines survival under zinc overload in a CUP1-null background. Its overexpression prevents the deleterious effects exhibited by CUP1-CRS5-null cells when exposed to combined Zn/Cu, as it does the mouse MT1 Zn-thionein, but not Cup1. The detailed characterization of Crs5 in vivo and in vitro Zn(II)-, Cd(II)- and Cu(I)-binding abilities fully supports its resemblance to mammalian MTs. Hence, Crs5 exhibits a good divalent metal-binding ability, yielding homometallic, highly chiral and stable Zn and Cd complexes when expressed in media enriched with these metal ions. In Cu-supplemented cultures, heterometallic Zn,Cu complexes are recovered, unless aeration is kept to a minimum. These features define a Crs5 dual metal-binding behaviour that is significantly closer to Zn-thioneins than to Cu-thioneins. Protein sequence similarities fully support these findings. Overall, a Crs5 function in global metal cell homeostasis, based on its Zn-binding features, is glimpsed. The comparative evaluation of Crs5 in the framework of MT functional differentiation and evolution allows its consideration as a representative of the primeval eukaryotic forms that progressively evolved to give rise to the Zn-thionein lineage.